P A Sharp

Massachusetts Institute of Technology, Cambridge, Massachusetts, United States

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Publications (300)4087.77 Total impact

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    ABSTRACT: CRISPR-Cas9 is a versatile genome editing technology for studying the functions of genetic elements. To broadly enable the application of Cas9 in vivo, we established a Cre-dependent Cas9 knockin mouse. We demonstrated in vivo as well as ex vivo genome editing using adeno-associated virus (AAV)-, lentivirus-, or particle-mediated delivery of guide RNA in neurons, immune cells, and endothelial cells. Using these mice, we simultaneously modeled the dynamics of KRAS, p53, and LKB1, the top three significantly mutated genes in lung adenocarcinoma. Delivery of a single AAV vector in the lung generated loss-of-function mutations in p53 and Lkb1, as well as homology-directed repair-mediated Kras(G12D) mutations, leading to macroscopic tumors of adenocarcinoma pathology. Together, these results suggest that Cas9 mice empower a wide range of biological and disease modeling applications.
    Cell. 09/2014;
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    ABSTRACT: The study of cancer genes in mouse models has traditionally relied on genetically-engineered strains made via transgenesis or gene targeting in embryonic stem cells. Here we describe a new method of cancer model generation using the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system in vivo in wild-type mice. We used hydrodynamic injection to deliver a CRISPR plasmid DNA expressing Cas9 and single guide RNAs (sgRNAs) to the liver that directly target the tumour suppressor genes Pten (ref. 5) and p53 (also known as TP53 and Trp53) (ref. 6), alone and in combination. CRISPR-mediated Pten mutation led to elevated Akt phosphorylation and lipid accumulation in hepatocytes, phenocopying the effects of deletion of the gene using Cre-LoxP technology. Simultaneous targeting of Pten and p53 induced liver tumours that mimicked those caused by Cre-loxP-mediated deletion of Pten and p53. DNA sequencing of liver and tumour tissue revealed insertion or deletion mutations of the tumour suppressor genes, including bi-allelic mutations of both Pten and p53 in tumours. Furthermore, co-injection of Cas9 plasmids harbouring sgRNAs targeting the β-catenin gene and a single-stranded DNA oligonucleotide donor carrying activating point mutations led to the generation of hepatocytes with nuclear localization of β-catenin. This study demonstrates the feasibility of direct mutation of tumour suppressor genes and oncogenes in the liver using the CRISPR/Cas system, which presents a new avenue for rapid development of liver cancer models and functional genomics.
    Nature 08/2014; · 38.60 Impact Factor
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    ABSTRACT: The p53-regulated long noncoding RNA lincRNA-p21 has been proposed to act in trans via several mechanisms ranging from repressing genes in the p53 transcriptional network to regulating mRNA translation and protein stability. To further examine lincRNA-p21 function, we generated a conditional knockout mouse model. We find that lincRNA-p21 predominantly functions in cis to activate expression of its neighboring gene, p21. Mechanistically, we show that lincRNA-p21 acts in concert with hnRNP-K as a coactivator for p53-dependent p21 transcription. Additional phenotypes of lincRNA-p21 deficiency could be attributed to diminished p21 levels, including deregulated expression and altered chromatin state of some Polycomb target genes, a defective G1/S checkpoint, increased proliferation rates, and enhanced reprogramming efficiency. These findings indicate that lincRNA-p21 affects global gene expression and influences the p53 tumor suppressor pathway by acting in cis as a locus-restricted coactivator for p53-mediated p21 expression.
    Molecular cell. 05/2014;
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    ABSTRACT: Specific protein-RNA interactions guide posttranscriptional gene regulation. Here, we describe RNA Bind-n-Seq (RBNS), a method that comprehensively characterizes sequence and structural specificity of RNA binding proteins (RBPs), and its application to the developmental alternative splicing factors RBFOX2, CELF1/CUGBP1, and MBNL1. For each factor, we recovered both canonical motifs and additional near-optimal binding motifs. RNA secondary structure inhibits binding of RBFOX2 and CELF1, while MBNL1 favors unpaired Us but tolerates C/G pairing in motifs containing UGC and/or GCU. Dissociation constants calculated from RBNS data using a novel algorithm correlated highly with values measured by surface plasmon resonance. Motifs identified by RBNS were conserved, were bound and active in vivo, and distinguished the subset of motifs enriched by CLIP-Seq that had regulatory activity. Together, our data demonstrate that RBNS complements crosslinking-based methods and show that in vivo binding and activity of these splicing factors is driven largely by intrinsic RNA affinity.
    Molecular cell. 05/2014;
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    ABSTRACT: MicroRNAs delicately regulate the balance of angiogenesis. Here we show that depletion of all microRNAs suppresses tumor angiogenesis. We generated microRNA-deficient tumors by knocking out Dicer1. These tumors are highly hypoxic but poorly vascularized, suggestive of deficient angiogenesis signaling. Expression profiling revealed that angiogenesis genes were significantly down-regulated as a result of the microRNA deficiency. Factor inhibiting hypoxia-inducible factor 1 (HIF-1), FIH1, is derepressed under these conditions and suppresses HIF transcription. Knocking out FIH1 using CRISPR/Cas9-mediated genome engineering reversed the phenotypes of microRNA-deficient cells in HIF transcriptional activity, VEGF production, tumor hypoxia, and tumor angiogenesis. Using multiplexed CRISPR/Cas9, we deleted regions in FIH1 3' untranslated regions (UTRs) that contain microRNA-binding sites, which derepresses FIH1 protein and represses hypoxia response. These data suggest that microRNAs promote tumor responses to hypoxia and angiogenesis by repressing FIH1.
    Genes & development 05/2014; · 12.08 Impact Factor
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    ABSTRACT: Bacterial type II CRISPR-Cas9 systems have been widely adapted for RNA-guided genome editing and transcription regulation in eukaryotic cells, yet their in vivo target specificity is poorly understood. Here we mapped genome-wide binding sites of a catalytically inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with single guide RNAs (sgRNAs) in mouse embryonic stem cells (mESCs). Each of the four sgRNAs we tested targets dCas9 to between tens and thousands of genomic sites, frequently characterized by a 5-nucleotide seed region in the sgRNA and an NGG protospacer adjacent motif (PAM). Chromatin inaccessibility decreases dCas9 binding to other sites with matching seed sequences; thus 70% of off-target sites are associated with genes. Targeted sequencing of 295 dCas9 binding sites in mESCs transfected with catalytically active Cas9 identified only one site mutated above background levels. We propose a two-state model for Cas9 binding and cleavage, in which a seed match triggers binding but extensive pairing with target DNA is required for cleavage.
    Nature Biotechnology 04/2014; · 32.44 Impact Factor
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    ABSTRACT: We demonstrate CRISPR-Cas9-mediated correction of a Fah mutation in hepatocytes in a mouse model of the human disease hereditary tyrosinemia. Delivery of components of the CRISPR-Cas9 system by hydrodynamic injection resulted in initial expression of the wild-type Fah protein in ∼1/250 liver cells. Expansion of Fah-positive hepatocytes rescued the body weight loss phenotype. Our study indicates that CRISPR-Cas9-mediated genome editing is possible in adult animals and has potential for correction of human genetic diseases.
    Nature Biotechnology 03/2014; · 32.44 Impact Factor
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    ABSTRACT: The tight regulation of splicing networks is critical for organismal development. To maintain robust splicing patterns, many splicing factors autoregulate their expression through alternative splicing-coupled nonsense-mediated decay (AS-NMD). However, as negative autoregulation results in a self-limiting window of splicing factor expression, it is unknown how variations in steady-state protein levels can arise in different physiological contexts. Here, we demonstrate that Rbfox2 cross-regulates AS-NMD events within RNA-binding proteins to alter their expression. Using individual nucleotide-resolution cross-linking immunoprecipitation coupled to high-throughput sequencing (iCLIP) and mRNA sequencing, we identified >200 AS-NMD splicing events that are bound by Rbfox2 in mouse embryonic stem cells. These "silent" events are characterized by minimal apparent splicing changes but appreciable changes in gene expression upon Rbfox2 knockdown due to degradation of the NMD-inducing isoform. Nearly 70 of these AS-NMD events fall within genes encoding RNA-binding proteins, many of which are autoregulated. As with the coding splicing events that we found to be regulated by Rbfox2, silent splicing events are evolutionarily conserved and frequently contain the Rbfox2 consensus UGCAUG. Our findings uncover an unexpectedly broad and multilayer regulatory network controlled by Rbfox2 and offer an explanation for how autoregulatory splicing networks are tuned.
    Genes & development 03/2014; 28(6):637-51. · 12.08 Impact Factor
  • Jesse R Zamudio, Timothy J Kelly, Phillip A Sharp
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    ABSTRACT: Argonaute (Ago) proteins mediate posttranscriptional gene repression by binding guide miRNAs to regulate targeted RNAs. To confidently assess Ago-bound small RNAs, we adapted a mouse embryonic stem cell system to express a single epitope-tagged Ago protein family member in an inducible manner. Here, we report the small RNA profile of Ago-deficient cells and show that Ago-dependent stability is a common feature of mammalian miRNAs. Using this criteria and immunopurification, we identified an Ago-dependent class of noncanonical miRNAs derived from protein-coding gene promoters, which we name transcriptional start site miRNAs (TSS-miRNAs). A subset of promoter-proximal RNA polymerase II (RNAPII) complexes produces hairpin RNAs that are processed in a DiGeorge syndrome critical region gene 8 (Dgcr8)/Drosha-independent but Dicer-dependent manner. TSS-miRNA activity is detectable from endogenous levels and following overexpression of mRNA constructs. Finally, we present evidence of differential expression and conservation in humans, suggesting important roles in gene regulation.
    Cell 02/2014; 156(5):920-34. · 31.96 Impact Factor
  • Xuebing Wu, Phillip A Sharp
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    ABSTRACT: The mammalian genome is extensively transcribed, a large fraction of which is divergent transcription from promoters and enhancers that is tightly coupled with active gene transcription. Here, we propose that divergent transcription may shape the evolution of the genome by new gene origination.
    Cell 11/2013; 155(5):990-996. · 31.96 Impact Factor
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    ABSTRACT: Transcription of the mammalian genome is pervasive, but productive transcription outside of protein-coding genes is limited by unknown mechanisms. In particular, although RNA polymerase II (RNAPII) initiates divergently from most active gene promoters, productive elongation occurs primarily in the sense-coding direction. Here we show in mouse embryonic stem cells that asymmetric sequence determinants flanking gene transcription start sites control promoter directionality by regulating promoter-proximal cleavage and polyadenylation. We find that upstream antisense RNAs are cleaved and polyadenylated at poly(A) sites (PASs) shortly after initiation. De novo motif analysis shows PAS signals and U1 small nuclear ribonucleoprotein (snRNP) recognition sites to be the most depleted and enriched sequences, respectively, in the sense direction relative to the upstream antisense direction. These U1 snRNP sites and PAS sites are progressively gained and lost, respectively, at the 5' end of coding genes during vertebrate evolution. Functional disruption of U1 snRNP activity results in a dramatic increase in promoter-proximal cleavage events in the sense direction with slight increases in the antisense direction. These data suggest that a U1-PAS axis characterized by low U1 snRNP recognition and a high density of PASs in the upstream antisense region reinforces promoter directionality by promoting early termination in upstream antisense regions, whereas proximal sense PAS signals are suppressed by U1 snRNP. We propose that the U1-PAS axis limits pervasive transcription throughout the genome.
    Nature 06/2013; · 38.60 Impact Factor
  • Jeremy E Wilusz, Phillip A Sharp
    Science 04/2013; 340(6131):440-1. · 31.20 Impact Factor
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    ABSTRACT: MicroRNAs (miRNAs) are critical to proliferation, differentiation, and development. Here, we characterize gene expression in murine Dicer-null adult mesenchymal stem cell lines, a fibroblast cell type. Loss of Dicer leads to derepression of let-7 targets at levels that exceed 10-fold to 100-fold with increases in transcription. Direct and indirect targets of this miRNA belong to a mid-gestation embryonic program that encompasses known oncofetal genes as well as oncogenes not previously associated with an embryonic state. Surprisingly, this mid-gestation program represents a distinct period that occurs between the pluripotent state of the inner cell mass at embryonic day 3.5 (E3.5) and the induction of let-7 upon differentiation at E10.5. Within this mid-gestation program, we characterize the let-7 target Nr6a1, an embryonic transcriptional repressor that regulates gene expression in adult fibroblasts following miRNA loss. In total, let-7 is required for the continual suppression of embryonic gene expression in adult cells, a mechanism that may underlie its tumor-suppressive function.
    Genes & development 04/2013; 27(8):941-54. · 12.08 Impact Factor
  • Allan M Gurtan, Phillip A Sharp
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    ABSTRACT: MicroRNAs (miRNAs) are key regulators of gene expression. They are conserved across species, expressed across cell types, and active against a large proportion of the transcriptome. The sequence-complementary mechanism of miRNA activity exploits combinatorial diversity, a property conducive to network-wide regulation of gene expression, and functional evidence supporting this hypothesized systems-level role has steadily begun to accumulate. The emerging models are exciting and will yield deep insight into the regulatory architecture of biology. However, because of the technical challenges facing the network-based study of miRNAs, many gaps remain. Here, we review mammalian miRNAs by describing recent advances in understanding their molecular activity and network-wide function.
    Journal of Molecular Biology 03/2013; · 3.91 Impact Factor
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    ABSTRACT: Many long noncoding RNA (lncRNA) species have been identified in mammalian cells, but the genomic origin and regulation of these molecules in individual cell types is poorly understood. We have generated catalogs of lncRNA species expressed in human and murine embryonic stem cells and mapped their genomic origin. A surprisingly large fraction of these transcripts (>60%) originate from divergent transcription at promoters of active protein-coding genes. The divergently transcribed lncRNA/mRNA gene pairs exhibit coordinated changes in transcription when embryonic stem cells are differentiated into endoderm. Our results reveal that transcription of most lncRNA genes is coordinated with transcription of protein-coding genes.
    Proceedings of the National Academy of Sciences 02/2013; · 9.81 Impact Factor
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    Anthony K L Leung, Phillip A Sharp
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    ABSTRACT: MicroRNAs (miRNAs) are a class of ∼22nt non-coding RNAs that regulate the translational potential and stability of mRNAs. Though constituting only 1-4% of human genes, miRNAs are predicted to regulate more than 60% of all mRNAs. The action of miRNAs is mediated through their associations with Argonaute proteins and mRNA targets. Previous studies indicated that though the majority of Argonaute proteins is diffusely distributed in the cytoplasm, a small fraction is consistently observed to be concentrated in a cytoplasmic compartment called GW/P-bodies. In this chapter, we will provide a quantitative and dynamic view of the subcellular localization of miRNA function, followed by a discussion on the possible roles of PBs in miRNA silencing.
    Advances in experimental medicine and biology 01/2013; 768:165-82. · 1.83 Impact Factor
  • Jeremy E. Wilusz, Phillip A. Sharp
    Science 01/2013; 340(6131):440-441. · 31.03 Impact Factor
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    ABSTRACT: The MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) locus is misregulated in many human cancers and produces an abundant long nuclear-retained noncoding RNA. Despite being transcribed by RNA polymerase II, the 3' end of MALAT1 is produced not by canonical cleavage/polyadenylation but instead by recognition and cleavage of a tRNA-like structure by RNase P. Mature MALAT1 thus lacks a poly(A) tail yet is expressed at a level higher than many protein-coding genes in vivo. Here we show that the 3' ends of MALAT1 and the MEN β long noncoding RNAs are protected from 3'-5' exonucleases by highly conserved triple helical structures. Surprisingly, when these structures are placed downstream from an ORF, the transcript is efficiently translated in vivo despite the lack of a poly(A) tail. The triple helix therefore also functions as a translational enhancer, and mutations in this region separate this translation activity from simple effects on RNA stability or transport. We further found that a transcript ending in a triple helix is efficiently repressed by microRNAs in vivo, arguing against a major role for the poly(A) tail in microRNA-mediated silencing. These results provide new insights into how transcripts that lack poly(A) tails are stabilized and regulated and suggest that RNA triple-helical structures likely have key regulatory functions in vivo.
    Genes & development 10/2012; · 12.08 Impact Factor
  • Phillip A Sharp
    Nature medicine 08/2012; 18(8):1169. · 27.14 Impact Factor
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    ABSTRACT: MicroRNAs are a class of short ~22 nucleotide RNAs predicted to regulate nearly half of all protein coding genes, including many involved in basal cellular processes and organismal development. Although a global reduction in miRNAs is commonly observed in various human tumors, complete loss has not been documented, suggesting an essential function for miRNAs in tumorigenesis. Here we present the finding that transformed or immortalized Dicer1 null somatic cells can be isolated readily in vitro, maintain the characteristics of DICER1-expressing controls and remain stably proliferative. Furthermore, Dicer1 null cells from a sarcoma cell line, though depleted of miRNAs, are competent for tumor formation. Hence, miRNA levels in cancer may be maintained in vivo by a complex stabilizing selection in the intratumoral environment.
    Cancer cell 06/2012; 21(6):848-55. · 25.29 Impact Factor

Publication Stats

34k Citations
4,087.77 Total Impact Points

Institutions

  • 1977–2014
    • Massachusetts Institute of Technology
      • • Department of Biology
      • • Koch Institute for Integrative Cancer Research
      Cambridge, Massachusetts, United States
  • 2013
    • Johns Hopkins University
      • Department of Biochemistry and Molecular Biology
      Baltimore, MD, United States
  • 1988–2013
    • Whitehead Institute for Biomedical Research
      • Department of Biology
      Cambridge, Massachusetts, United States
    • University of Adelaide
      Tarndarnya, South Australia, Australia
  • 2000
    • University of Massachusetts Medical School
      • Department of Biochemistry and Molecular Pharmacology
      Worcester, MA, United States
  • 1999
    • Tokyo Institute of Technology
      Edo, Tōkyō, Japan
  • 1998
    • University of Kentucky
      Lexington, Kentucky, United States
  • 1995
    • National Institutes of Health
      • Laboratory of Viral Diseases
      Bethesda, MD, United States
  • 1993
    • Aarhus University
      Aarhus, Central Jutland, Denmark
  • 1989
    • Fred Hutchinson Cancer Research Center
      Seattle, Washington, United States